Recent advances in biological fluid analysis, and in particular, analytical hematology have increased the quantity and quality of information available from a patients blood sample. As a result, the medical community's interest in using patients blood samples as a diagnostic tool has also increased, and the most common test that is performed on anticoagulated whole blood is the complete blood count, or CBC, which is a suite of tests which are considered to include measurements of the hematocrit (Hct), hemoglobin (Hgb), red blood cell count (RBC). white blood cell count (WBC) and platelet count (Plt), red blood cell metrics such as the mean cell volume (MCV) and others, as well as the leukocyte differential count (LDC or "Diff") which is the classification of the types of white blood cells present. Compared to any other laboratory test, it is a peculiar characteristic of the CBC, that any instrument or method which performs it must do four different types of analyses. First, the general physical properties of the sample, namely the hematocrit and various cell or particle counts must be analyzed using quantitative methods relating to the entire sample. In conventional instrumentation and methods, this requires accurate sample metering and dilution, followed by specialized measurement apparatus. Secondly, a specific chemical property of the sample, namely the hemoglobin concentration, must be measured, usually by quantitative chemical means. Thirdly, the instrument must measure quantitative aspects of the individual cells, which usually involves providing a high dilution of the sample with a subsequent passage of the diluted material through a flow cell which measures the cells using electrical or optical means. Fourthly, qualitative measurements are used to classify the percentage of the total white blood cells which are composed of specific sub-populations. The number of sub-populations depends upon the sophistication of the instrument involved, which may be as little as two or more than seven classifications.
Historically, the different aspects of the CBC have been performed using separate methods. For example, the LDC portion of a CBC was traditionally performed by smearing a small amount of undilute blood on a slide, staining it, and examining the smear under a microscope. Reasonable results can be gained from such a smear, but the accuracy and reliability of the data depends largely on the technician's experience and technique. In addition, the use of blood smears is labor intensive and cost prohibitive, and is therefore generally not favored for commercial applications. Another method uses electrical impedance or optical flow cytometry. Flow cytometry involves passing a diluted blood sample through a small vessel wherein electrical impedance or optical sensors can evaluate the constituent cells as they pass serially through the vessel. The same apparatus may also be used to simultaneously enumerate and provide cell metric data. To evaluate WBC's and/or platelets, the blood sample must be diluted, and the sample must be treated to mitigate the overwhelming number of the RBC's relative to the WBC's and the platelets. Although more expedient and consistent than the above described smear methods, flow cytometry also possesses several disadvantages. One disadvantage of flow cytometry is the plumbing and fluid controls that are necessary for controlling the flow rate of the diluted blood sample past the sensors, The plumbing in current flow cytometers can, and often does, leak, thus potentially compromising the accuracy and the safety of the equipment. Another disadvantage of many current flow cytometers relates to the accuracy of the internal fluid flow controls and automated dilution equipment. The accuracy of the flow cytometer depends upon the accuracy of the fluid flow controls and the sample dilution equipment, and their ability to remain accurately calibrated. Flow controls and dilution equipment require periodic recalibration. The need for recalibration illustrates the potential for inaccurate results and the undesirable operating costs that exist with many presently available flow cytometers. An article authored by John L. Haynes, and published in Cytometry Supplement 3: 7-17 in 1988 describes the principles of flow cytometry, both impedance and optical, and the application of such a technology to various fields of endeavor. Blood samples being examined in flow cytometers are diluted anywhere from 10:1 to 50,000:1.
Another approach to cellular analysis is volumetric capillary scanning as outlined in U.S. Pat. Nos. 5,547,849; 5,585,246 and others, wherein a relatively undiluted sample of whole blood is placed into a capillary of known volume and thickness and is examined while the blood is in a quiescent state. This technique deals with the presence of the red blood cells by limiting the scanning wavelengths to those to which the red blood cells are relatively transparent, and it requires that the sample be treated so that the red blood cells do not aggregate during the measurement process. Thus, this technique is limited to the use of longer wavelength fluorescence, and there is no provision for the examination of red blood cells and platelets or the examination of any cellular morphology. Also, because the counts must occur in a constant volume, it is difficult or impossible to examine a wide range of sample particulate constituents in a single sample vessel, since the relative numbers of these constituents can vary over a thousand to one in a whole blood sample. There are a number of commercial instruments available for performing a CBC or related tests, but those which provide more than a few of the CBC tests quickly become complex, expensive and prone to malfunction. In addition, there are a number of methods proposed for specific hematological tests, but these do not provide all of the clinically useful information which is expected in a CBC.
Another problem with the more complex currently available instruments for performing CBC's is that they must be calibrated. This is because most of the dilutions and measurements are relative rather than absolute, so in order to provide exact quantitation, actual particulates, generally stabilized samples of whole blood with known values, must be analyzed by the instruments, and the instrument adjusted so that the correct values are produced. It should be obvious that this type of calibration is prone to errors in the preparation of the standard material and its stability during transportation and storage. The standard material is also expensive, which increases the cost of the tests.
In a co-pending U.S. patent application Ser. No. 09/249,721, a method is disclosed which allows the measurement of many important blood parameters within a quiescent layer of substantially undiluted whole blood. An important feature of the said invention is the lack of need for any external calibration material, but to be optimally accurate, it requires a means of accurately ensuring the dimensional accuracy of the chambers of the described device.
It would be desirable to have a method and apparatus for examining a quiescent sample of anticoagulated whole blood, which method and apparatus are capable of providing accurate qualitative and quantitative results on a number of different hematologic parameters, and does not require sample fluid flow through the sampling chamber during sample analysis. It would be desirable to provide such a method and apparatus which could derive volumetric blood cell counts and cell volume information from the quiescent blood sample and did not require external materials for its calibration.